J Life Sci Res 2016; 14:1-4Ⓒ 2016 Osaka Prefecture University

Journal of Life Science Research
1Online ISSN 2186-5809

Rapid Communication

Preparation of TPP+-selective membrane electrodes using polar aprotic solvents
Makoto Muratsugu,† Momoko Okabe, and Sae Inagake
Bioanalytical Science Laboratory, Department of Clinical Nutrition, Osaka Prefecture University, Habikino, Osaka 583-8555, Japan.
Received 15 September 2016; accepted 12 November 2016

To embed TPP-TPB complex in PVC membrane, DMA and DMF were the most suitable polar aprotic solvents (among DMA, DMF, DMSO, and THF) that we examined. TPP＋-selective electrodes prepared with
TPP-TPB-containing membranes showed linearity from 10－7 to 10－2 mol L－1 TPP＋. The slopes of the TPP＋
-selective electrodes were a little lower than the theoretical value, but we think that the electrodes could bepractically used to measure TPP＋ concentration. The life spans of the TPP＋-selective electrodes made with
the TPP-TPB-containing membranes were longer than that of the TPP＋-selective electrode made with TPB－containing membrane.
Key words: tetraphenyl phosphonium (TPP+); polar aprotic solvent; TPP+-selective membrane electrode; membrane potential

1 Introduction
The difference in electrical potential across the cell
membrane of a living cell plays an absolutely essential
role in its physiological functions.1-5 The membrane potential of a large cell like a giant nerve cell can be measured using a microelectrode method, but those of small
cells like bacteria and organella like mitochondria cannot
be measured using this method. Fluorescent probes6-9 orradiolabeled probes10-14 are widely used to measure themembrane potentials of bacteria and mitochondria. Selective electrodes for lipid-soluble cations (unlabeled probes;
TPP＋ 15-17 and DDA＋ 18,19) that are considered to permeate
freely to biological membranes have been developed, andthe change in the membrane potential of small vesicles
can be continuously and easily monitored with these electrodes. DDA＋ requires catalytic amounts of TPB− to penetrate to biological membrane,20 but TPP＋ does not.
PVC-based membranes sensitive to DDA＋ and TPP＋
are lipid-soluble cations containing TPB− as an ion-exchanger. THF is a good solvent for PVC; it dissolvedDDA-TPB complex, and the complex was easily embedded in PVC membrane.19 However, TPP-TPB complex ishighly insoluble in THF, one of the polar aprotic solvents,†Corresponding author, Email: mmakoto@rehab.osakafu-u.ac.jp

Abbreviations: TPP＋, tetraphenyl phosphonium; DDA＋, dibenzyl dimethylammonium; TPB－, tetraphenylborate; PVC, polyvinyl chloride; THF,tetrahydrofuran; DMA, N,N-dimethylacetamide; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide

and it was hard to embed that complex in PVC membrane.15, 16
We examined whether we could use other polar
aprotic solvents (DMA, DMF, and DMSO) to dissolve
TPP-TPB complex. As far as we know, TPP-TPB complex was successfully embedded into PVC membrane forthe first time. We investigated the properties (responseand life span) of TPP＋-selective electrodes made using
PVC membrane containing TPP-TPB complex.
2 Materials and Methods
2.1 Materials
The following materials were obtained from the indicated sources: N,N-dimethylacetamide (DMA), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO),and tetraphenylphosphonium tetraphenylborate (TPPTPB) from Wako Pure Chemical Industries, Ltd., Osaka,
Japan; polyvinyl chloride (PVC), and dioctyl phthalate(DOP) from Sigma-Aldrich, Co., St. Louis, MO, U.S.A.;
tetraphenylphosphonium chloride (TPPCl) from Alfa Aesar, Heysham, United Kingdom; tetraphenylborate sodium(TPBNa) from Dojindo Labs, Kumamoto, Japan; tetrahydrofuran (THF) from Nacalai Tesque, Inc., Kyoto, Japan;
agar from Difco Labs., Detroit, U.S.A.
All other chemicals were of reagent grade or better.
The water used was 17-Mohm grade.
2.2 Selection of a solvent suitable for membrane preparation
0.1 g of PVC was added to 2 mL of DMA, DMF,

2DMSO, or THF. 6.58 mg of TPP-TPB was added to 1 mLof DMA, DMF DMSO, or THF. The mixtures were vigorously stirred using a stirrer.
2.3 Preparation of PVC membrane including TPP-TPB
complex or TPB−
0.6, 1.2, 2.4, or 3.6 mL of 10−2 mol L−1 TPP-TPB in
DMA or DMF was added to each flat petri dish (φ5.5 cm),then 0.57 mL of DOP was added to the petri dishes, and3.8 mL of 5 % PVC in THF was finally added followed bythorough stirring to ensure uniformity. The solvent wasthen slowly evaporated at room temperature. For the sakeof simplicity, the membrane prepared with 0.6 mL of
10−2 mol L−1 TPP-TPB in DMA was called as DMA(0.6)
membrane, for example.
0.6, 1.2, 2.4, or 3.6 mL of 10−2 mol L−1 TPB− in THF
was added to each flat petri dish (φ 5.5 cm), then 0.57 mLof DOP was added to the petri dishes, and 3.8 mL of 5 %PVC in THF was finally added followed by thorough stirring to ensure uniformity. The solvent was then slowly
evaporated at room temperature. For the sake of simplicity, the membrane prepared with 0.6 mL of 10−2 mol L−1
TPB− in THF was called THF(0.6) membrane, for example.
2.4 Construction and properties of TPP＋-selective membrane electrode
A piece of each of the membranes containing TPPTPB or TPB− was glued to a PVC tube (inner diameter0.85 cm) with THF. The PVC tube with the membrane
containing TPB− was soaked overnight in 10−2 mol L−1
TPP＋ for conditioning before use.
A 10−2 mol L−1 TPP＋-solution (aqueous) was placed
inside the PVC tube as an internal reference solution.
This solution and a calomel electrode was bridged with asmall polyethylene tube (φ 2 mm) filled with saturated
KCl agar (a salt bridge). This device was referred to as
the TPP＋-selective membrane electrode or TPP＋-electrode.
The TPP＋-electrodes prepared with DMA(0.6), (1.2),(2.4), or (3.6) membrane contained 0.93, 1.9, 3.7, or 5.6
μmol of TPP-TPB complex, respectively. The TPP＋-electrodes prepared with DMF (0.6), (1.2), (2.4), or (3.6)
membrane contained 0.93, 1.9, 3.7, or 5.6 μmol of TPPTPB complex, respectively. The TPP＋-electrodes prepared with THF(0.6), (1.2), (2.4), or (3.6) membrane contained 0.93, 1.9, 3.7, or 5.6 μmol of TPB−, respectively.
For the sake of simplicity, the electrodes containing 0.93μmol TPP-TPB complex were referred to as a DMA(0.93)
or DMF(0.93) electrode, depending on the kind of solvent
used, and so on. The electrode containing 0.93 μmol
TPB− was referred to as THF(0.93) electrode.
The electromotive force between the TPP＋-electrodeand the calomel reference electrode in the sample solution

Fig. 1 onstruction of TPP＋-electrode and diagram ofC
apparatus for measuring Δ| E|.
CE, calomel electrode.

was measured by an electrometer (TR 8651, Takeda Riken, Tokyo, Japan) connected to a pen-writing recorder(R300RV, Rika-Denki, Tokyo, Japan) (Fig. 1).
We kept the DMA(5.6) and DMF(5.6) electrodes inpurified water and the THF(3.7) electrode in 10−2 mol L−1
TPP＋ to examine the life spans of these electrodes. Wewashed the electrodes with purified water and removed
the water attached to the electrodes before measurement.
3 Results and Discussion
3.1 Selection of a solvent suitable for membrane preparation
We tried to dissolve TPP-TPB complex in DMA,DMF, DMSO, and THF. We used THF to reconfirm thatthe complex was insoluble in THF. The complex could bedissolved to 10−2 mol L−1 in DMA and DMF, but it was insoluble at 10−1 mol L−1 in DMA and DMF by visual observation. The complex was insoluble in DMSO and THF byvisual observation. PVC was insoluble in DMSO by visual observation, but dissolved in THF, DMA, and DMF.
5 % PVC in THF, DMA, and DMF were placed in petri
dishes and were allowed to stand overnight at room temperature. THF completely evaporated and a thin flattransparent PVC membrane formed. Evaporation of DMA
and DMF were incomplete, with a cloudy PVC membrane
and a little solvent remaining in the petri dishes. Thus, weused DMA or DMF to dissolve TPP-TPB complex andTHF was used as the solvent for PVC.
3.2 Properties of the electrodes
The electrical potential of a TPP＋-electrode is givenby the Nernst equation,

3(1)where E°, R, T, F, and [TPP＋] are the standard potential,the gas constant (8.32 J mol−1 K−1), the temperature of thesystem in kelvins (K), the Faraday constant (9.65 × 104 Cmol−1), and the concentration of TPP＋, respectively. The
difference of electrical potential, |ΔE| between E1 at [TPP＋]1and E2 at [TPP＋]2 is given by the following equation,(2)
If [TPP＋]1 is 10[TPP＋]2 in Eq. (2), |ΔE| is 58 mV at 20 C.
Fig. 2 shows the response of each TPP＋-electrodefrom 10−8 to 10−2 mol L−1 TPP＋ (aqueous solution). The
properties of each TPP＋-electrode from these data are summarized in Table 1. The theoretical value of the slope was
58 mV between 20 and 22 C. The slopes of DMA( * )and DMF( * ) electrodes increased with increasing amountof TPP-TPB complex (ion-exchanger), and were almost
constant for the electrodes with more than 3.7 μmol of
TPP-TPB complex. The slopes of DMA(3.7), DMA(5.6),
DMF(3.7), and DMF(5.6) electrodes were about 50 mV
and were a little lower than the theoretical value (58 mV).
However, DMA(3.7), DMA(5.6), DMF(3.7), andDMF(5.6) electrodes could be used to measure the TPP＋concentration. The slopes of THF(0.93), THF(1.9), andTHF(3.7) electrodes were close to the theoretical value regardless of the TPB− amount included in the membrane.
The slope of THF(5.6) electrode was somewhat higher
than the theoretical value. It is unknown at present why
the slopes of THF( * ) electrodes did not increase with increasing amount of TPB− in the PVC membrane. Thiswill be the subject of a future study.
We examined the life spans of DMA(5.6), DMF(5.6),
and THF(3.7) electrodes. Fig. 3 shows the change overtime for the slopes of these electrodes. For this examination we used THF(3.7) electrode having a slope close tothat of DMA(5.6), and DMF(5.6) electrode instead ofTable 1Fig. 2

Properties of the TPP＋-electrodes.

Response of the TPP＋-electrodes to TPP＋ solutions (aqueous) of varying concentration.
(a) □, DMA(0.93) electrode; ■, DMA(1.9) electrode; ○, DMA(3.7) electrode; ●, DMA(5.6) electrode.
(b) □, DMF(0.93) electrode; ■, DMF(1.9) electrode; ○, DMF(3.7) electrode; ●, DMF(5.6) electrode.
(c) □, THF(0.93) electrode; ■, THF(1.9) electrode; ○, THF(3.7) electrode; ●, THF(5.6) electrode.

Fig. 3

Life span of TPP＋-electrodes.

 , DMA(5.6) electrode; ■, DMF(5.6) electrode;●○, THF(3.7) electrode.
The dashed line is a theoretical value.

4THF(5.6) electrode. The slopes of DMA(5.6) andDMF(5.6) electrodes were constant for about 200 days after preparation, but the slope of THF(3.7) electrode wasreduced by about 55 % at 175 days after preparation.
We consider that DMA(5.6) and DMF(5.6) electrodes
can be used as a device for measuring membrane potential
in biological systems, in place of the TPP＋-electrodes21, 22
used in recent years. However, we must also examine
substances that may interfere with these electrodes, depending on the biological system where the electrodes areused, and selectivity coefficients23 must be evaluated forthese electrodes in biological systems.

12131415

Conflict of Interest
All the authors declare that they have no conflict ofinterest.
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